Seroprevalence and risk factors of brucellosis in Arabian horses

Abstract Background Brucellosis, as a zoonotic disease, mainly occurs in horses by Brucella abortus, Brucella canis and Brucella suis. The disease in equines is often asymptomatic, but the clinical signs in horses are mostly characterized by bursitis, arthritis and tenosynovitis. Objectives This study, thus, aimed to determine the seroprevalence of brucellosis and its associated risk factors in the Arabian horses of Khuzestan province, South‐west Iran. Methods To that end, the blood samples randomly collected from 180 Arabian horses were analyzed for the presence of anti‐Brucella antibodies by Rose Bengal plate test (RBPT), serum agglutination test (SAT), 2‐mercaptoethanol test (2‐ME) and a commercial i‐ELISA kit. Results The ROC curve analysis showed that the best cut‐off point for S/P values in i‐ELISA turned out to be 26.25%. The results showed that the overall seroprevalence of brucellosis based on parallel interpretation of the test results was 12.22% (Positive/Tested = 22/180). The prevalence of acute and chronic brucellosis was 8.3 and 3.9%, respectively. The seroprevalence of brucellosis with RBPT and i‐ELISA methods was 1.11% (2/180) and 7.22% (13/180), respectively. According to what SAT revealed, 9.44% (17/180) of sera had a titer of 40 or greater, and at 2‐ME, 7.22% of samples (13 out of 180 samples) depicted a titer of 40. The results of i‐ELISA, SAT and 2‐ME were significantly different from those of RBPT (p < 0.01); however, there was no significant difference between i‐ELISA, SAT and 2‐ME in findings (p > 0.05). Conclusions The results of this study recommend that i‐ELISA be used for screening purposes of brucellosis in horses. The findings confirmed that Arabian horses are natural hosts for the Brucellae. It is, thus, necessary to adopt appropriate prevention and control programs by health authorities and horse owners so as to reduce the distribution and transmission of the infection in the regions where brucellosis is prevalent.


INTRODUCTION
Brucellosis is one of the most important zoonotic diseases with a global distribution that affects many humans and animals (Corbel, 1997). Equine brucellosis is caused by Brucella abortus, Brucella suis and Brucella canis and the natural infection may be caused through ingestion of infected material, respiratory system or skin wounds (Lucero et al., 2008). Many horses enter a latent infection state following infection, and despite having positive agglutination titer, they do not show clinical symptoms. However, non-specific symptoms such as weakness, depression, muscle stiffness, intermittent fever and movement disorders are seen in some horses infected with Brucella.
Furthermore, brucellosis in equines may be associated with fistulous withers, inflammation of the atlantal bursa, carpal bursitis, tenosynovitis, osteomyelitis, osteoarthritis and rarely reproductive disorders (Cohn et al., 1992;Cvetnic et al., 2005;Ocholi et al., 2004). Despite the high cost of controlling brucellosis, as an endemic disease in Iran, through vaccination, testing and slaughtering of domestic ruminants, this disease has been seen in all parts of the country for more than half a century, and thousands of people are infected each year, as well.
The average incidence of brucellosis in the Iranian human population was 21 cases per 100,000 populations, although this varied between 1.5 and 107.5 per 100,000 population in different parts of the country (Zeinali et al., 2011). Also, the serological prevalence of equine brucellosis in some parts of Iran varied from 0 to 12% (Badiei et al., 2013;Gharekhani et al., 2020;Ghobadi & Salehi, 2013;Hashemitabar & Poursafar, 2005;Nemati, 2017;Rafeiei Sharebabaki, 2018;Tahamtan et al., 2008Tahamtan et al., , 2010Taheri, 2018 (Antunes et al., 2013;Ducrotoy et al., 2018;Esmaeili, 2014;Hussain et al., 2020;Tel et al., 2011). However, among these serological methods, the SAT is able to detect the total amount of agglutinin antibodies, including IgM and IgG (mainly IgM), whereas 2-ME and i-ELISA measure IgG antibodies (Godfroid, et al., 2010;Roushan et al., 2010).  (Esmaeili, 2014). Thus, the epidemiological study of brucellosis in the Arabian horses is important because in addition to its pathogenicity for horses, it can be transmitted from horses to humans and other animals. Certainly, to prevent and, in particular, eradicate a disease, its agent hosts should be considered.
Brucellae are no exception to the rule. Therefore, in the present study, in addition to determining the prevalence of brucellosis infection in the Arabian horses in the southwest of Iran through serological methods, including RBPT, SAT, 2-ME and i-ELISA, the serological methods used were also evaluated and the associated risk factors affecting the infection were identified, as well.

Study area and population
This epidemiological study was carried out in Khuzestan Province located in the southwest of Iran (Figure 1). Province varies from arid to humid and its northern parts experience cold weather, whereas its southern parts experience tropical weather (Zarasvandi et al., 2011). Therefore, to create regional differences in the epidemiological determinants, such as environment and manage-

Serological analysis
All 180 collected serum samples were evaluated for anti-Brucella antibodies by RBPT, SAT (Wright test) and 2-ME using whole cell antigen (Razi Vaccine and Serum Research Institute, Iran) according to OIE manual, and a commercial indirect IgG ELISA test (ID vet, France, ID Screen Brucellosis Serum Indirect Multi-species). The SAT was considered positive when titer was at least 40 (Alton et al., 1988;Denny, 1972;Tel et al., 2011;Yilmaz & Wilson, 2013). The ID vet ELISA kit was initially designed to diagnose brucellosis in cattle, sheep, goats and pigs, and there was no information on the possibility of using it for the diagnosis of horse brucellosis. Therefore, to use i-ELISA kit in the present study, its efficiency for the diagnosis of brucellosis in horses was initially analyzed by several equine sero-positive and sero-negative samples. Having confirmed that i-ELISA kit was also suitable for the detection of anti-Brucella antibodies in horse serum samples, we calculated its appropriate cut-off for diagnosis of equine brucellosis. For this purpose, 90 equine sera (70 Wright-sero-negative and 20 sero-positive) were assessed according to the kit manufacturer's instructions. The optical density of samples (OD Sample ), kits positive (OD PC ) and negative controls (OD NC ) were recorded and the S/P% was calculated for each sample according to the following formula: The cut-off point of the i-ELISA kit for the diagnosis of equine brucellosis was calculated by ROC (receiver operating characteristic) curve analysis.

Statistical analysis
Statistical analysis of data was performed using SPSS (version 22.0; SPSS for Windows Inc., Chicago, Illinois). The association between age (1-2 years, 3-9 years or ≥ 10 years), sex (male or female), history of leaving the region (yes or no), body condition score (good or bad), type TA B L E 1 Absolute frequency of positive samples (+) based on Rose Bengal plate test (RBPT), serum agglutination test (SAT), 2-mercaptoethanol test (2-ME) and i-ELISA

Seroprevalence of Brucella
The overall seroprevalence of brucellosis (acute and chronic) was

Evaluation of serological methods
The Cochran's Q test showed that there was no significant difference between diagnostic methods including RBPT, SAT, 2-ME and i-ELISA (Cochran's Q = 16.82, df = 3, p = 0.001). A comparison of the 2-ME and SAT tests showed that all negative cases in SAT were negative in 2-ME and also many positive cases in the SAT were also positive in 2-ME ( Table 2). Comparison of the 2-ME and i-ELISA showed that many positive cases in 2-ME were negative in i-ELISA and also many positive cases in i-ELISA were negative in 2-ME, but many negative cases in the 2-ME were also negative in i-ELISA (Table 3) showed that the 2-ME did not differ significantly from the ID vet ELISA and SAT in the diagnosis of equine brucellosis (p > 0.05). Comparison of the i-ELISA and SAT showed that many positive cases in SAT were negative in i-ELISA and also some positive cases in i-ELISA were negative in SAT (Table 4). The McNemar test showed that the SAT did not differ significantly from the ID vet ELISA in the diagnosis of equine brucellosis (p > 0.05).
Comparison of the SAT and 2-ME with RBPT revealed that all positive cases in the SAT or 2-ME were negative in the RBPT. Besides, it was found that all cases that were positive in the RBPT were not detectable in the SAT or 2-ME, but many negative cases in the SAT or 2-ME were also negative in RBPT. The results of RBPT were significantly different from the SAT (p < 0.001) and 2-ME (p = 0.007) in the diagnosis of equine brucellosis. Generally, there was a significant difference between the two methods (p = 0.001). A comparison of the RBPT and i-ELISA tests showed that all positive cases in RBPT were negative in ID vet ELISA, and all positive cases in ID vet ELISA were negative in RBPT. Rose Bengal was significantly different from the ID vet ELISA (p = 0.007).

The role of associated factors
To investigate the risk factors affecting the seroprevalence of brucellosis at the horse level, the overall seroprevalence of brucellosis based on parallel interpretation of the test results was used and the strength of association of them is summerized in Table 5

DISCUSSION
In this epidemiological study, the seroprevalence of brucellosis was determined in Arabian horses in south-western Iran (Khuzestan Province). Brucellosis is a worldwide zoonosis posing heavy economic and public health damage. The results of this study could be a reflection of brucellosis seroprevalence in other livestock and humans in the region, and would determine the role of Arabian horses in disease epidemiology. The findings can also help health policymakers make better decisions to control and prevent the disease.
Serological techniques can pinpoint the potent humoral immune responses, triggered by exposure to Brucella. In such cases, the emergence of IgG antibodies occurs far later than IgM. Such antibodies do not disappear even after the response reaches its highest point (3-4 weeks post-infection) and thus, can be identified over longer duration of time (up to several years); quite conversely, despite the swift induction of IgM antibodies (2-3 weeks after exposure), they linger merely for a few months (Godfroid et al., 2002;Saegerman et al., 2004;Sutherland, 1984). What differentiates acute infections from chronic ones is the kinetics of production, the emergence of the principal immunoglobulin isotypes over the course of the infection, and the function of these immunoglobulins in various serological trials. Although the simultaneous presence of IgM (detected in an agglutination test) and IgG (detected in i-ELISA and 2-ME) signifies acute brucellosis, the sole presence of IgG characterizes the chronic brucellosis (Godfroid et al., 2010).
In this study, the prevalence of acute brucellosis was relatively higher than the chronic form (8.3 vs. 3.9%). However, the horses featured no clinical signs, such as weakness, depression, muscle stiffness, intermittent fever, fistulous withers, reproductive disorders and movement disorders. Under multi-species housing in Khuzestan province, horses are grazed, watered and kept in a close contact with cattle, sheep and goats. Therefore, horses usually become infected through ingesting the Brucella-contaminated feed and water, and they would show the subclinical and asymptomatic forms of the infection.
There was also a significant difference between the serological diagnostic methods used in terms of findings. As such, all of the positive cases in RBPT were negative in SAT, 2-ME and i-ELISA tests, possibly due to non-specific reactions in RBPT (Young, 1991). In addition, many of the positive cases in the SAT were negative in i-ELISA, indicating that there were some non-specific reactions in SAT as com-  (Lewis, et al., 2008). Consequently, false-negative results may arise if these commercial kits are deployed for studying infectious diseases in certain animals. In this study, a num-ber of samples turned out negative for i-ELISA test despite demonstrating the IgG titer in Wright and 2-ME-Wright tests. In this research, no statistically significant differences were defined between the SAT, 2-ME and i-ELISA results; this issue indicated the higher level of the IgG than IgM in the tested serums. However, the non-significant difference between of the mentioned tests, suggested existence of dissimilar percent of the specific anti-brucella IgG sub-classes in the infected animals.
IgG sub-classes do not have the same tendency to agglutinate the bacteria or bind to the conjugated anti-globulin antibodies.
Ardo and Abubakar (2016)  Therefore, the use of kits made in European countries is not recommended and it is suggested that their cut-off points be recalculated and localized before use, as it was done in the present study. As such, the best cut-off point for the kits used was determined by the ROC curve.
Although the presence of anti-Brucella antibodies disclosed the exposure to Brucella spp., the exact Brucella species responsible for inducing the secretion of those antibodies remains unclear due to the limitations the current study faced with. In addition, due to the vulnerability of nearly all animal species to Brucella infection and consequently, the loss of antibody titres, there exists a high likelihood that the actual prevalence of brucellosis is shown higher than that unveiled by antibody detection. It is noteworthy that a positive result yielded by the agglutination test, chiefly utilized for the identification of IgM, cannot confirm brucellosis if it is not substantiated by i-ELISA within 1 week, an obstacle the present cross sectional study (parallel testing) failed to overcome (Godfroid, et al., 2010).

CONCLUSIONS
This epidemiological study showed that the Brucella infection is relatively prevalent (about 10%) in Arabian horses in the southwest of Iran and confirmed that they are the natural hosts of the Brucellae, although the infection is latent or sub-clinical in them. Even though, the role of horses in transmitting Brucella to other animals is less important, they can play a significant role in the epidemiology of the disease as a reservoir or secondary host to preserve this bacterium. The results showed that the sensitivity of i-ELISA was more than RBPT, so it is recommended that i-ELISA be used for screening purposes of brucellosis in Arabian horse population due to its short time required to perform, relatively simple and standardized implementation, measuring a large number of samples simultaneously and cost-effectiveness.
Accordingly, the role of horse as an intervening factor in the control of brucellosis in human should be carefully considered by healthcare providers. Given the limited number of literature in this field, the findings of the present study can be helpful in conducting other epidemiological studies and controlling programs.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.759